Evaluating the In-vitro Antibacterial Effect of Iranian Propolis on Oral Microorganisms

Propolis has traditionally been used in curing infections and healing wounds and burns. Current researches have shown that propolis has antibacterial, antifungal and antiviral actions however, the pharmacological activity of propolis is highly variable depending on its geographic origin. There have been few studies on the effects of Iranian propolis on the oral microorganisms. In this in-vitro study, the antimicrobial activity of the ethanolic and water extracts of the Iranian propolis (10%, w/v) from north-east area of Tehran was evaluated. Susceptibility of the oral strains tested (Streptococcus mutans ATCC 35668; Streptococcus salivarius ATCC 9222; Staphylococcus aureus ATCC 25923; Enterococcus faecalis ATCC 9854 and Lactobacillus casei ATCC 39392) was evaluated using the agar diffusion method at a concentration of 20 mg/mL of propolis and the zones of growth inhibition were measured. Antibacterial activity was determined by using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) at different concentrations of propolis. The ethanolic extract showed bacteriostatic and bactericidal activity against all the strains, with MIC and MBC ranges of 250-500 µg/mL. The MIC concentration of the water extract was 500 µg/mL against S. mutans and E. faecalis. The water extract showed bactericidal activity only against S. mutans (20 mg/mL). These results indicate that the ethanolic extract is probably more useful in the control of oral biofilms and subsequent dental caries development. However, to determine the consequence of the ethanolic extract of Iranian propolis on the oral mucosa, in-vivo studies of its possible effects are needed.


Introduction
Propolis is a natural non-toxic beehive product, which is used for building and restoration of the honeycomb (1). The term propolis comes from the Greek 'pro', in front, 'polis' means 'town' or 'city' and bees use propolis to seal their hives against the attack of the other insects (2). In the hive, propolis act as a biocide, being active against the invasive bacteria, fungi and even invading larvae (3). Other biological activities have also been depicted for propolis, including antibacterial (4), antifungal (5), antiviral (6), antitumor (7), immunomodulation (8) and antiactivity of propolis ethanolic extract of different geographic origin against oral pathogens has been studied by several authors (4, 10-12); however, few studies have investigated on Iranian propolis (13-16).
on the tooth surface (17), plays an important role in the etiology of dental caries and periodontal disease (18). Thus, the control of dental plaque is one of the targets for the prevention of dental caries and periodontal disease. Several different approaches have been proposed for controlling agents that not only reduce the viability, but also control the colonization and accumulation of cariogenic bacteria on the tooth surface could be more effective. microorganisms in the oral cavity (20). However, the use of chlorhexidine, as an anti-caries agent, not only remains controversial (21) but also has common side effects, including the formation of extrinsic stain on the tooth and tongue (22). As a result, there is a huge interest in the plants and natural product have been studied for their potential in the prevention of dental caries (21, 23). Despite increasing the use of propolis worldwide, only a few studies have been carried out to determine the inhibitory effect of Iranian propolis against some bacteria of relevance in dentistry (24,25). Within this context, the aim of this study was to evaluate the antibacterial activity of the ethanolic and water extracts of Iranian propolis against several oral pathogens.

Experimental
Extraction of propolis Propolis sample was collected from colonies of honeybees located in the north-east area of Tehran in Iran. Hand collected propolis was kept in a dry place and stored at 4 C until its complete process. The sample was chopped into small blender and extracted with 80% ethanol (1 : 10 w/v) in a shaker at room temperature for 48 h. The ethanolic extract solution (EEP) was then concentrated in a rotary evaporator (Heidolph, Germany) to obtain the crude extract in paste form and kept in a dry and dark place.
For obtaining water extract of propolis (WEP), with distilled water (1 : 10 w/v) by means of continuous stirring at room temperature for 48 h.
a rotary evaporator to afford crude extract. Propolis crude extracts were redissolved in 80% ethanol or distilled water at a concentration used for antibacterial testing. Antimicrobial activity Antimicrobial activity of Iranian propolis extracts was investigated using agar diffusion method. Test plates (diameter 10 cm) were wells of 8 mm in diameter were punched in the agar plates by using sterile glass-made pipettes attached to a vacuum pump. Sterile swabs were dipped into the bacterial suspension containing 1.5 × 10 8 the extracts or negative controls (80% ethanol and distilled water). Two wells without the extracts served as the positive control. The plates were kept for 2 h at room temperature to allow the diffusion of the agents through the agar. Afterwards, the plates were incubated at for an appropriate period of time (aerobes, 24 h and L.Casei, 48 h in an anaerobic jar). Zones of inhibition of microbial growth around the holes were measured and recorded after the incubation time. The inhibitory zone was considered the shortest distance (mm) from the outside margin of the samples to the initial point of the microbial growth. All measurements were performed twice by the same blinded operator. Five replicates were made for each microorganism.

Effects on viability of suspension cells
The MIC was determined based on the macrodilution tube methods (TSB or MRS broth for L. Casei the determination of MIC, inoculum suspensions were prepared from 24 h broth cultures. Diluted propolis diluted with the liquid medium to reach 6 fold dilutions. There were also control tubes with the liquid medium (without propolis) as negative controls and 80% ethanol and distilled water as lowest concentration that restricted the bacterial growth to an absorbance lower than 0.05 at 550 nm (invisible growth).
For the determination of MBC, Sterile swabs were dipped into the tube that contained propolis concentrations higher than the MIC and inoculated onto the agar medium. The MBC was no visible growth on the agar.

Statistical analysis
The results were summarized as mean ± standard deviation and analyzed with SPSS (Version 16.0). The data were submitted to analysis of variance using ANOVA test. The Inhibition zone values are given in mm (mean ± SD; n = 5). Negative control was inactive.

Results and Discussion
As part of a continuing study on the prevention of dental diseases by natural drugs, we hypothesized that Iranian propolis, may be a valuable resource for the control of the development.
antibacterial activity of the extracts using the agar-well diffusion method. Table 1 present the mean diameters of growth inhibition zones' values (mm) obtained for each tested strain. The ethanolic extract of propolis (EEP) produced inhibitory zones against all the tested microorganisms. Among the strains tested with EEP, the most sensitive was S. salivarius, which showed the highest inhibition zones (20.00 ± 1.00 mm). The most resistant strain was L. Casei with growth inhibition zones of 12.00 ± 1.00 mm. Growth of the S.mutans and Staph. aureus previous results (11,26). On the other hand, the water extract of Iranian propolis demonstrated only slight activity, in which 3 out of 5 strains presented no zone of growth inhibition. WEP showed slight activity only against S. mutans and E. faecalis. d The results showed that, at effective than WEP (p < 0.05). The negative controls (water and ethanol) did not show any inhibitory effects on the tested microorganisms. The MICs and MBCs of the test substances are shown in Tables 2 and 3 for all strains. MIC was determined as the lowest concentration of the propolis extract, which inhibited the growth of the tested microorganisms ( Table 2). The EEP showed a MIC and MBC in the range of 250-S. mutans and E. faecalis and did not show any bactericidal activity at the concentrations used in this assay, with the exception of S. mutans The applied studies on the antimicrobial activity of propolis show different results (27).
of propolis could be due to the differences in its chemical components (27). It has also been reported that the samples collected from different geographic origin with different climates and vegetation show different antibacterial activities (28). Moreover, seasonal effect on Brazilian propolis antibacterial activity has been investigated with other researchers (29).
The Iranian propolis extract used in this study showed proper antibacterial capacity, however, it